The present invention relates to monoclonal antibodies recognizing antigens expressed on the surface of tumor vessel endothelial cells, hybridomas producing said antibodies, pharmaceutical agents comprising said antibodies, as well as pharmaceutical or diagnostic agents comprising a conjugate of one of said antibodies and another conjugating molecule.
Currently known anticancer agents include anticancer DDS agents using antibodies against tumor cells as a targeting carrier. However, this type of anticancer agent has the disadvantage that tumor interstitial pressure or other barriers hinder permeation of the agent to significantly lower the performance of drug delivery to the target. Moreover, a particular antibody could not be used as a common targeting carrier for various types of tumors, because of heterogeneity of antigens on the surface of tumor cells.
However, there may be expressed common specific molecules in tumor tissue vessel endothelial cells (TEC) irrespective of the type of cancer because tumor vessels have many common characteristics such as enhanced permeation function. Targeting therapy directed to tumor vessels is expected to effectively achieve the advantages of anticancer DDS agents without concern about barriers to transfer to tissues, and therefore, monoclonal antibodies which are specific for vessel endothelial cells, particularly vessel endothelial cells existing in tumor tissues (tumor vessel endothelial cells) have been developed.
Up to the present, markers widely recognizing vessel endothelial cells such as CD31, CD36, Ulex europaeus-I agglutinin (UEA-1) and markers generally expressed in activated large vessel endothelial cells such as von Willebrand factor (vWF), ICAM-1 (CD54), E-selectin have been known.
On the other hand, tumor vessel endothelial cells are postulated to express specific antigens which are not expressed in normal tissues as described above, and various antibodies recognizing tumor vessel endothelial cells have been reported up to the present, among which some examples are listed below:
E-9 antibody (Wang, J. M. et al., Int. J. Cancer (1993) 54, 363);
anti-FB5 (endosialin) antibody (Rettig, W. J. Pro. Natl. Sci. USA (1992) 89, 10832);
H4/18 antibody (Cotran, R. S. et al., J. Exp. Med. (1986) 164, 661);
Q BEND/10 antibody (Ramani, P. et al., Histopathology (1990) 17, 237);
EN4/EN3 antibody (Schlingemann, R. O. et al., Amer. J. Pathol. (1991) 138, 1335);
BMA120 antibody (Schlingemann, R. O. et al., Amer. J. Pathol. (1991) 138, 1335);
EN7/44 antibody (Hagemeier, H. H. et al., Int. J. Cancer (1986) 38, 481);
PAL-E antibody (Schlingemann, R. O. et al., Amer. J. Pathol. (1991) 138, 1335);
HEC-1 antibody (Gougos, A. et al., J. Immunol. (1988) 141, 1934);
TEC4 and TEC11 antibodies (Thorpe, P. E. et al., Am. Assoc. Cancer Res. (abstract) (1994) 35, 379).
However, antigens recognized by these antibodies are also expressed in normal endothelial cells or other cell species, though in a minor amount, and nothing has been reported about antitumor effects of these antibodies, leading to a demand for development of antibodies which are more suitable for the therapy of solid cancers targeting tumor vessel endothelial cells.
CD 44 is a known antigenic protein existing on the surface of lymphocytes or the like (J. Immunol., 142, 2045-2051, Mar. 15, 1989), and monoclonal antibodies against CD44 have been reported (JP Laid-open Publication (Kokai) No. 508309/93; WO94/12631; and WO95/33771). However, these antibodies against CD44 have not been reported to recognize antigens on the surface of tumor vessel endothelial cells, and CD44 has not been reported to exist on the surface of tumor vessel endothelial cells, either, prior to the priority date of the present application.
An object of the present invention is to provide novel monoclonal antibodies recognizing antigens on the surface of tumor vessel endothelial cells.
Another object of the present invention is to provide novel monoclonal antibodies recognizing antigens specifically existing on the surface of tumor vessel endothelial cells.
Still another object of the present invention is to provide hybridomas producing novel monoclonal antibodies recognizing antigens on the surface of tumor vessel endothelial cells.
Still another object of the present invention is to provide pharmaceutical agents comprising novel monoclonal antibodies recognizing antigens on the surface of tumor vessel endothelial cells.
Still another object of the present invention is to provide pharmaceutical or diagnostic agents comprising a conjugate of a monoclonal antibody recognizing an antigen on the surface of tumor vessel endothelial cells and a conjugating molecule for treating or diagnosing tumor-associated conditions.
As a result of careful studies to solve the above problems, the present inventors succeeded in obtaining monoclonal antibodies recognizing antigens existing on the surface of tumor vessel endothelial cells, analyzing the distribution of the obtained antibodies in organs and the antigenic proteins, and confirming that said monoclonal antibodies administered alone or in combination with other drugs to tumor-transplanted rats show inhibitory effects on solid cancers, and thus accomplished the present invention.
Accordingly, the present invention provides, in the broadest sense, monoclonal antibodies recognizing antigens on the surface of tumor vessel endothelial cells, hybridomas producing said monoclonal antibodies, pharmaceutical agents comprising said monoclonal antibodies, as well as pharmaceutical or diagnostic agents comprising a conjugate of one of said monoclonal antibodies and another conjugating molecule.
According to a first aspect of the present invention, a monoclonal antibody which recognizes an antigen of a molecular weight of 40 kD or 80 kD on the surface of tumor vessel endothelial cells is provided.
According to the present invention, said monoclonal antibody wherein the affinity for tumor vessel endothelial cells is comparable to or higher than the affinity for normal vessel endothelial cells when the affinity for tumor vessel endothelial cells and normal vessel endothelial cells both immobilized is measured by ELISA is provided.
Also according to the present invention, said monoclonal antibody wherein the reactivity to tumor tissues is comparable to or higher than the reactivity to the liver and kidney when the reactivity to tissue samples of tumor, liver and kidney of rats is measured by immunohistochemical staining is provided.
Also according to the present invention, said monoclonal antibody wherein the level in tumor tissues is comparable to or higher than the level in blood and the level in the liver, lung, spleen, small intestine and muscle is comparable to or lower than the level in blood when the antibody is administered to rats bearing tumors is provided.
Also according to the present invention, said monoclonal antibody produced by hybridomas of a single clone obtained by the steps of immunizing an animal with cell membrane vesicles prepared from tumor vessel endothelial cells from rat KMT-17 solid cancer, isolating antibody-producing cells from said animal, fusing said antibody-producing cells to myeloma cells to prepare hybridomas, and screening the thus-obtained hybridomas, is provided.
Also according to the present invention, said monoclonal antibody wherein said immunization procedure further comprises passively immunizing said animal with an antiserum against cell membrane vesicles prepared from normal vessel endothelial cells before immunizing said animal with cell membrane vesicles prepared from tumor vessel endothelial cells is provided.
Also according to the present invention, said monoclonal antibody produced by hybridomas bearing the accession number FERM BP-5786 or FERM BP-5787 is provided.
Also according to the present invention, said monoclonal antibody having antitumor activity is provided.
According to a second aspect of the present invention, a hybridoma producing said monoclonal antibody according to the present invention is provided.
Particularly preferably, a hybridoma bearing the accession number FERM BP-5786 or FERM BP-5787 is provided according to the present invention.
According to a third aspect of the present invention, a pharmaceutical agent comprising said monoclonal antibody is provided.
Preferably, said pharmaceutical agent for the treatment of tumors is provided according to the present invention.
According to a fourth aspect of the present invention, a pharmaceutical or diagnostic agent comprising a conjugate of (a) said monoclonal antibody and (b) a conjugating molecule selected from the group consisting of anticancer chemotherapeutic agents, radioisotopes, proteinous toxins, lethal proteins or expression vectors carrying genes expressing them, enzymes, streptavidin is provided.
Preferably, said pharmaceutical or diagnostic agent for the treatment or diagnosis of tumors is provided.